Temporal Enzymatic Treatment to Enhance the Remodeling of Multiple Cartilage Microtissues into a Structurally Organized Tissue.

Scaffold-free tissue engineering aims to recapitulate key aspects of normal developmental processes to generate biomimetic grafts. Although functional cartilaginous tissues are engineered using such approaches, considerable challenges remain. Herein, the benefits of engineering cartilage via the fusion of multiple cartilage microtissues compared to using (millions of) individual cells to generate a cartilaginous graft are demonstrated. Key advantages include the generation of a richer extracellular matrix, more hyaline-like cartilage phenotype, and superior shape fidelity. A major drawback of aggregate engineering is that individual microtissues do not completely (re)model and remnants of their initial architectures remain throughout the macrotissue. To address this, a temporal enzymatic (chondroitinase-ABC) treatment is implemented to accelerate structural (re)modeling and shown to support robust fusion between adjacent microtissues, enhance microtissue (re)modeling, and enable the development of a more biomimetic tissue with a zonally organized collagen network. Additionally, enzymatic treatment is shown to modulate matrix composition, tissue phenotype, and to a lesser extent, tissue mechanics. This work demonstrates that microtissue self-organization is an effective method for engineering scaled-up cartilage grafts with a predefined geometry and near-native levels of matrix accumulation. Importantly, key limitations associated with using biological building blocks can be alleviated by temporal enzymatic treatment during graft development.

Integrating Melt Electrowriting and Fused Deposition Modeling to Fabricate Hybrid Scaffolds Supportive of Accelerated Bone Regeneration.

Emerging additive manufacturing (AM) strategies can enable the engineering of hierarchal scaffold structures for guiding tissue regeneration. Here, the advantages of two AM approaches, melt electrowriting (MEW) and fused deposition modelling (FDM), are leveraged and integrated to fabricate hybrid scaffolds for large bone defect healing. MEW is used to fabricate a microfibrous core to guide bone healing, while FDM is used to fabricate a stiff outer shell for mechanical support, with constructs being coated with pro-osteogenic calcium phosphate (CaP) nano-needles. Compared to MEW scaffolds alone, hybrid scaffolds prevent soft tissue collapse into the defect region and support increased vascularization and higher levels of new bone formation 12 weeks post-implantation. In an additional group, hybrid scaffolds are also functionalized with BMP2 via binding to the CaP coating, which further accelerates healing and facilitates the complete bridging of defects after 12 weeks. Histological analyses demonstrate that such scaffolds support the formation of well-defined annular bone, with an open medullary cavity, smooth periosteal surface, and no evidence of abnormal ectopic bone formation. These results demonstrate the potential of integrating different AM approaches for the development of regenerative biomaterials, and in particular, demonstrate the enhanced bone healing outcomes possible with hybrid MEW-FDM constructs.

An assessment of the response of human MSCs to hydrostatic pressure in environments supportive of differential chondrogenesis.

Mechanical stimulation can modulate the chondrogenic differentiation of stem/progenitor cells and potentially benefit tissue engineering (TE) of functional articular cartilage (AC). Mechanical cues like hydrostatic pressure (HP) are often applied to cell-laden scaffolds, with little optimization of other key parameters (e.g. cell density, biomaterial properties) known to effect lineage commitment. In this study, we first sought to establish cell seeding densities and fibrin concentrations supportive of robust chondrogenesis of human mesenchymal stem cells (hMSCs). High cell densities (15*106 cells/ml) were more supportive of sGAG deposition on a per cell basis, while collagen deposition was higher at lower seeding densities (5*106 cells/ml). Employment of lower fibrin (2.5 %) concentration hydrogels supported more robust chondrogenesis of hMSCs, with higher collagen type II and lower collagen type X deposition compared to 5 % hydrogels. The application of HP to hMSCs maintained in identified chondro-inductive culture conditions had little effect on overall levels of cartilage-specific matrix production. However, if hMSCs were first temporally primed with TGF-ß3 before its withdrawal, they responded to HP by increased sGAG production. The response to HP in higher cell density cultures was also associated with a metabolic shift towards glycolysis, which has been linked with a mature chondrocyte-like phenotype. These results suggest that mechanical stimulation may not be necessary to engineer functional AC grafts using hMSCs if other culture conditions have been optimised. However, such bioreactor systems can potentially be employed to better understand how engineered tissues respond to mechanical loading in vivo once removed from in vitro culture environments.

A microphysiological model of bone development and regeneration.

Endochondral ossification (EO) is an essential biological process than underpins how human bones develop, grow, and heal in the event of a fracture. So much is unknown about this process, thus clinical manifestations of dysregulated EO cannot be adequately treated. This can be partially attributed to the absence of predictivein vitromodels of musculoskeletal tissue development and healing, which are integral to the development and preclinical evaluation of novel therapeutics. Microphysiological systems, or organ-on-chip devices, are advancedin vitromodels designed for improved biological relevance compared to traditionalin vitroculture models. Here we develop a microphysiological model of vascular invasion into developing/regenerating bone, thereby mimicking the process of EO. This is achieved by integrating endothelial cells and organoids mimicking different stages of endochondral bone development within a microfluidic chip. This microphysiological model is able to recreate key events in EO, such as the changing angiogenic profile of a maturing cartilage analogue, and vascular induced expression of the pluripotent transcription factors SOX2 and OCT4 in the cartilage analogue. This system represents an advancedin vitroplatform to further EO research, and may also serve as a modular unit to monitor drug responses on such processes as part of a multi-organ system.

Engineering High-Quality Cartilage Microtissues Using Hydrocortisone Functionalized Microwells.

Engineering clinically relevant musculoskeletal tissues at a human scale is a considerable challenge. Developmentally inspired scaffold-free approaches for engineering cartilage tissues have shown great promise in recent years, enabling the generation of highly biomimetic tissues. Despite the relative success of these approaches, the absence of a supporting scaffold or hydrogel creates challenges in the development of large-scale tissues. Combining numerous scaled-down tissue units (herein termed microtissues) into a larger macrotissue represents a promising strategy to address this challenge. The overall success of such approaches, however, relies on the development of strategies which support the robust and consistent chondrogenic differentiation of clinically relevant cell sources such as mesenchymal stem/stromal cells (MSCs) within microwell arrays to biofabricate numerous microtissues rich in cartilage-specific extracellular matrix components. In this article, we first describe a simple method to manufacture cartilage microtissues at various scales using novel microwell array stamps. This system allows the rapid and reliable generation of cartilage microtissues and can be used as a platform to study microtissue phenotype and development. Based on the unexpected discovery that Endothelial Growth Medium (EGM) enhanced MSC aggregation and chondrogenic capacity within the microwell arrays, this work also sought to identify soluble factors within the media capable of supporting robust differentiation using heterogeneous MSC populations. Hydrocortisone was found to be the key factor within EGM that enhanced the chondrogenic capacity of MSCs within these microwell arrays. This strategy represents a promising means of generating large numbers of high-quality, scaffold-free cartilage microtissues for diverse biofabrication applications. Impact statement This study addresses a key challenge facing emerging modular biofabrication strategies that use microtissues as biological building blocks. Namely, achieving the necessary robust and consistent differentiation of clinically relevant cell sources, for example, mesenchymal stem/stromal cells (MSCs), and the accumulation of sufficient tissue-specific extracellular matrix (ECM) to engineer tissue of scale. We achieved this by establishing hydrocortisone as a simple and potent method for improving MSC chondrogenesis, resulting in the biofabrication of high-quality (ECM rich) cartilage microtissues. These findings could enable the generation of more scalable engineered cartilage by ensuring the formation of high-quality microtissue building blocks generated using heterogeneous MSC populations.

Spatial patterning of phenotypically distinct microtissues to engineer osteochondral grafts for biological joint resurfacing.

Modular biofabrication strategies using microtissues or organoids as biological building blocks have great potential for engineering replacement tissues and organs at scale. Here we describe the development of a biofabrication strategy to engineer osteochondral tissues by spatially localising phenotypically distinct cartilage microtissues within an instructive 3D printed polymer framework. We first demonstrate that immature cartilage microtissues can spontaneously fuse to form homogeneous macrotissues, and that combining less cellular microtissues results in superior fusion and the generation of a more hyaline-like cartilage containing higher levels of sulphated glycosaminoglycans and type II collagen. Furthermore, temporally exposing developing microtissues to transforming growth factor-ß accelerates their volumetric growth and subsequent capacity to fuse into larger hyaline cartilage grafts. Next, 3D printed polymeric frameworks are used to further guide microtissue fusion and the subsequent self-organisation process, resulting in the development of a macroscale tissue with zonal collagen organisation analogous to the structure seen in native articular cartilage. To engineer osteochondral grafts, hypertrophic cartilage microtissues are engineered as bone precursor tissues and spatially localised below phenotypically stable cartilage microtissues. Implantation of these engineered grafts into critically-sized caprine osteochondral defects results in effective defect stabilisation and histologically supports the restoration of a more normal articular surface after 6 months in vivo. These findings support the use of such modular biofabrication strategies for biological joint resurfacing.

Scaffold microarchitecture regulates angiogenesis and the regeneration of large bone defects.

Emerging 3D printing technologies can provide exquisite control over the external shape and internal architecture of scaffolds and tissue engineering (TE) constructs, enabling systematic studies to explore how geometric design features influence the regenerative process. Here we used fused deposition modelling (FDM) and melt electrowriting (MEW) to investigate how scaffold microarchitecture influences the healing of large bone defects. FDM was used to fabricate scaffolds with relatively large fibre diameters and low porosities, while MEW was used to fabricate scaffolds with smaller fibre diameters and higher porosities, with both scaffolds being designed to have comparable surface areas. Scaffold microarchitecture significantly influenced the healing response following implantation into critically sized femoral defects in rats, with the FDM scaffolds supporting the formation of larger bone spicules through its pores, while the MEW scaffolds supported the formation of a more round bone front during healing. After 12 weeksin vivo, both MEW and FDM scaffolds supported significantly higher levels of defect vascularisation compared to empty controls, while the MEW scaffolds supported higher levels of new bone formation. Somewhat surprisingly, this superior healing in the MEW group did not correlate with higher levels of angiogenesis, with the FDM scaffold supporting greater total vessel formation and the formation of larger vessels, while the MEW scaffold promoted the formation of a dense microvasculature with minimal evidence of larger vessels infiltrating the defect region. To conclude, the small fibre diameter, high porosity and high specific surface area of the MEW scaffold proved beneficial for osteogenesis and bone regeneration, demonstrating that changes in scaffold architecture enabled by this additive manufacturing technique can dramatically modulate angiogenesis and tissue regeneration without the need for complex exogenous growth factors. These results provide a valuable insight into the importance of 3D printed scaffold architecture when developing new bone TE strategies.

Promoting endogenous articular cartilage regeneration using extracellular matrix scaffolds.

Articular cartilage defects fail to heal spontaneously, typically progressing to osteoarthritis. Bone marrow stimulation techniques such as microfracture (MFX) are the current surgical standard of care; however MFX typically produces an inferior fibro-cartilaginous tissue which provides only temporary symptomatic relief. Here we implanted solubilised articular cartilage extracellular matrix (ECM) derived scaffolds into critically sized chondral defects in goats, securely anchoring these implants to the joint surface using a 3D-printed fixation device that overcame the need for sutures or glues. In vitro these ECM scaffolds were found to be inherently chondro-inductive, while in vivo they promoted superior articular cartilage regeneration compared to microfracture. In an attempt to further improve the quality of repair, we loaded these scaffolds with a known chemotactic factor, transforming growth factor (TGF)-ß3. In vivo such TGF-ß3 loaded scaffolds promoted superior articular cartilage regeneration. This study demonstrates that ECM derived biomaterials, either alone and particularly when combined with exogenous growth factors, can successfully treat articular cartilage defects in a clinically relevant large animal model.

Bilayered extracellular matrix derived scaffolds with anisotropic pore architecture guide tissue organization during osteochondral defect repair.

While some clinical advances in cartilage repair have occurred, osteochondral (OC) defect repair remains a significant challenge, with current scaffold-based approaches failing to recapitulate the complex, hierarchical structure of native articular cartilage (AC). To address this need, we fabricated bilayered extracellular matrix (ECM)-derived scaffolds with aligned pore architectures. By modifying the freeze-drying kinetics and controlling the direction of heat transfer during freezing, it was possible to produce anisotropic scaffolds with larger pores which supported homogenous cellular infiltration and improved sulfated glycosaminoglycan deposition. Neo-tissue organization in vitro could also be controlled by altering scaffold pore architecture, with collagen fibres aligning parallel to the long-axis of the pores within scaffolds containing aligned pore networks. Furthermore, we used in vitro and in vivo assays to demonstrate that AC and bone ECM derived scaffolds could preferentially direct the differentiation of mesenchymal stromal cells (MSCs) towards either a chondrogenic or osteogenic lineage respectively, enabling the development of bilayered ECM scaffolds capable of spatially supporting unique tissue phenotypes. Finally, we implanted these scaffolds into a large animal model of OC defect repair. After 6 months in vivo, scaffold implantation was found to improve cartilage matrix deposition, with collagen fibres preferentially aligning parallel to the long axis of the scaffold pores, resulting in a repair tissue that structurally and compositionally was more hyaline-like in nature. These results demonstrate how scaffold architecture and composition can be spatially modulated to direct the regeneration of complex interfaces such as the osteochondral unit, enabling their use as cell-free, off-the-shelf implants for joint regeneration. STATEMENT OF SIGNIFICANCE: The architecture of the extracellular matrix, while integral to tissue function, is often neglected in the design and evaluation of regenerative biomaterials. In this study we developed a bilayered scaffold for osteochondral defect repair consisting of tissue-specific extracellular matrix (ECM)-derived biomaterials to spatially direct stem/progenitor cell differentiation, with a tailored pore microarchitecture to promote the development of a repair tissue that recapitulates the hierarchical structure of native AC. The use of this bilayered scaffold resulted in improved tissue repair outcomes in a large animal model, specifically the ability to guide neo-tissue organization and therefore recapitulate key aspects of the zonal structure of native articular cartilage. These bilayer scaffolds have the potential to become a new therapeutic option for osteochondral defect repair.

Gremlin-1 Suppresses Hypertrophy of Engineered Cartilage In Vitro but Not Bone Formation In Vivo.

Current repair of articular cartilage (AC) often leads to a lower quality tissue with an unstable hypertrophic phenotype, susceptible to endochondral ossification and development of osteoarthritis. Engineering phenotypically stable AC remains a significant challenge in the cartilage engineering field. This motivates new strategies inspired from the extracellular matrix proteins unique to phenotypically stable AC. We have previously shown that the bone morphogenetic protein antagonist gremlin-1 (GREM1) protein, present in permanent but not transient cartilage, suppresses the hypertrophy of chondrogenically primed bone marrow stem cells (BMSCs) in pellet culture. The goal of this study was to assess the effect of GREM1 on the in vitro and in vivo phenotypic stability of porcine BMSC-derived cartilage engineered within chondro-permissive scaffolds. In addition, we explored whether GREM1 would synergize with physioxia, a potent chondrogenesis regulator, when engineering cartilage grafts. GREM1 did not influence the expression of chondrogenic markers (SOX-9, COL2A1), but did suppress the expression of hypertrophic markers (MMP13, COL10A1) in vitro. Cartilage engineered with GREM1 contained higher levels of residual cartilage after 4 weeks in vivo, but endochondral bone formation was not prevented. Higher GREM1 levels did not significantly alter the fate of engineered tissues in vitro or in vivo. The combination of physioxia and GREM1 resulted in a higher sulfated glycosaminoglycan deposition in vitro and a greater retention of cartilage matrix in vivo than physioxia alone, but again did not suppress endochondral ossification. Therefore, while physioxia and GREM1 regulate BMSC chondrogenesis in vitro and reduce cartilage loss in vivo, their use does not guarantee the development of stable cartilage. Impact Statement A major challenge associated with bone marrow stem cell (BMSC)-derived cartilage is that the chondrocyte-like cells have a tendency to undergo hypertrophic differentiation, yielding tissue unsuitable for use in hyaline articular cartilage (AC) repair. This is motivating the development of new tissue engineering strategies to generate phenotypically stable chondrocyte-like cells from BMSCs. In this study, we aimed to engineer phenotypically stable cartilage grafts using BMSCs seeded onto solubilized AC extracellular matrix-derived scaffolds and treated with the bone morphogenetic protein antagonist gremlin-1. This article describes the effects of potential therapeutic strategies that could improve the phenotypic stability of chondrogenically differentiated BMSCs, and examined the use of this strategy both in vitro and in vivo.

A Spheroid Model of Early and Late-Stage Osteosarcoma Mimicking the Divergent Relationship between Tumor Elimination and Bone Regeneration.

Osteosarcoma is the most diagnosed bone tumor in children. The use of tissue engineering strategies after malignant tumor resection remains a subject of scientific controversy. As a result, there is limited research that focuses on bone regeneration postresection, which is further compromised following chemotherapy. This study aims to develop the first co-culture spheroid model for osteosarcoma, to understand the divergent relationship between tumor elimination and bone regeneration. By manipulating the ratio of stromal to osteosarcoma cells the modelled cancer state (early/late) is modified, as is evident by the increased tumor growth rates and an upregulation of a panel of well-established osteosarcoma prognostic genes. Validation of the authors' model is conducted by analyzing its ability to mimic the cytotoxic effects of the FDA-approved chemotherapeutic Doxorubicin. Next, the model is used to investigate what effect osteogenic supplements have, if any, on tumor growth. When their model is treated with osteogenic supplements, there is a stimulatory effect on the surrounding stromal cells. However, when treated with chemotherapeutics this stimulatory effect is significantly diminished. Together, the results of this study present a novel multicellular model of osteosarcoma and provide a unique platform for screening potential therapeutic options for osteosarcoma before conducting in vivo experiments.

Additive manufacturing of cartilage-mimetic scaffolds as off-the-shelf implants for joint regeneration.

Biomimetic scaffolds that provide a tissue-specific environment to cells are particularly promising for tissue engineering and regenerative medicine applications. The goal of this study was to integrate emerging additive manufacturing and biomaterial design strategies to produce articular cartilage (AC) mimetic scaffolds that could be used as 'off-the-shelf' implants for joint regeneration. To this end alginate sulfate, a sulfated glycosaminoglycan (sGAG) mimic, was used to functionalize porous alginate-based scaffolds and to support the sustained release of transforming growth factor-ß3 (TGF-ß3). Covalent crosslinking dramatically improved the elasticity of the alginate/alginate sulfate scaffolds, while scaffold architecture could be tailored using a directional freezing technique. Introducing such an anisotropic architecture was found to promote mesenchymal stem cell (MSC) infiltration into the scaffold and to direct the orientation of the deposited extracellular matrix, leading to the development of cartilage tissue with a biomimetic zonal architecture.In vitroexperiments also demonstrated the capacity of the sulfated scaffolds to both enhance chondrogenesis of MSCs and to control the release of TGF-ß3, leading to the development of a tissue rich in sGAG and type II collagen. The scaffolds were further reinforced with a 3D printed poly(lactide-co-ϵ-caprolactone) (PLCL) framework, leading to composite implants that were more elastic than those reinforced with polycaprolactone, and which better mimicked the bulk mechanical properties of native cartilage tissue. The ability of this composite scaffold to support chondrogenesis was then confirmed within a dynamic culture system. Altogether, these findings demonstrate the potential of such biomimetic scaffolds as putative 'single-stage' or 'off-the-shelf' strategies for AC regeneration.

Bioprinting of biomimetic self-organised cartilage with a supporting joint fixation device.

Despite sustained efforts, engineering truly biomimetic articular cartilage (AC) via traditional top-down approaches remains challenging. Emerging biofabrication strategies, from 3D bioprinting to scaffold-free approaches that leverage principles of cellular self-organisation, are generating significant interest in the field of cartilage tissue engineering as a means of developing biomimetic tissue analoguesin vitro.Although such strategies have advanced the quality of engineered cartilage, recapitulation of many key structural features of native AC, in particular a collagen network mimicking the tissue's 'Benninghoff arcade', remains elusive. Additionally, a complete solution to fixating engineered cartilagesin situwithin damaged synovial joints has yet to be identified. This study sought to address both of these key challenges by engineering biomimetic AC within a device designed to anchor the tissue within a synovial joint defect. We first designed and fabricated a fixation device capable of anchoring engineered cartilage into the subchondral bone. Next, we developed a strategy for inkjet printing porcine mesenchymal stem/stromal cells (MSCs) into this supporting fixation device, which was also designed to provide instructive cues to direct the self-organisation of MSC condensations towards a stratified engineered AC. We found that a higher starting cell-density supported the development of a more zonally defined collagen network within the engineered tissue. Dynamic culture was implemented to further enhance the quality of this engineered tissue, resulting in an approximate 3 fold increase in glycosaminoglycan and collagen accumulation. Ultimately this strategy supported the development of AC that exhibited near-native levels of glycosaminoglycan accumulation (>5% WW), as well as a biomimetic collagen network organisation with a perpendicular to a parallel fibre arrangement (relative to the tissue surface) from the deep to superficial zones via arcading fibres within the middle zone of the engineered tissue. Collectively, this work demonstrates the successful convergence of novel biofabrication methods, bioprinting strategies and culture regimes to engineer a hybrid implant suited to resurfacing AC defects.

Biofabrication of Prevascularised Hypertrophic Cartilage Microtissues for Bone Tissue Engineering.

Bone tissue engineering (TE) has the potential to transform the treatment of challenging musculoskeletal pathologies. To date, clinical translation of many traditional TE strategies has been impaired by poor vascularisation of the implant. Addressing such challenges has motivated research into developmentally inspired TE strategies, whereby implants mimicking earlier stages of a tissue's development are engineered in vitro and then implanted in vivo to fully mature into the adult tissue. The goal of this study was to engineer in vitro tissues mimicking the immediate developmental precursor to long bones, specifically a vascularised hypertrophic cartilage template, and to then assess the capacity of such a construct to support endochondral bone formation in vivo. To this end, we first developed a method for the generation of large numbers of hypertrophic cartilage microtissues using a microwell system, and encapsulated these microtissues into a fibrin-based hydrogel capable of supporting vasculogenesis by human umbilical vein endothelial cells (HUVECs). The microwells supported the formation of bone marrow derived stem/stromal cell (BMSC) aggregates and their differentiation toward a hypertrophic cartilage phenotype over 5 weeks of cultivation, as evident by the development of a matrix rich in sulphated glycosaminoglycan (sGAG), collagen types I, II, and X, and calcium. Prevascularisation of these microtissues, undertaken in vitro 1 week prior to implantation, enhanced their capacity to mineralise, with significantly higher levels of mineralised tissue observed within such implants after 4 weeks in vivo within an ectopic murine model for bone formation. It is also possible to integrate such microtissues into 3D bioprinting systems, thereby enabling the bioprinting of scaled-up, patient-specific prevascularised implants. Taken together, these results demonstrate the development of an effective strategy for prevascularising a tissue engineered construct comprised of multiple individual microtissue "building blocks," which could potentially be used in the treatment of challenging bone defects.

Printing New Bones: From Print-and-Implant Devices to Bioprinted Bone Organ Precursors.

Regenerating large bone defects remains a significant clinical challenge, motivating increased interest in additive manufacturing and 3D bioprinting to engineer superior bone graft substitutes. 3D bioprinting enables different biomaterials, cell types, and growth factors to be combined to develop patient-specific implants capable of directing functional bone regeneration. Current approaches to bioprinting such implants fall into one of two categories, each with their own advantages and limitations. First are those that can be 3D bioprinted and then directly implanted into the body and second those that require further in vitro culture after bioprinting to engineer more mature tissues prior to implantation. This review covers the key concepts, challenges, and applications of both strategies to regenerate damaged and diseased bone.

Affinity-bound growth factor within sulfated interpenetrating network bioinks for bioprinting cartilaginous tissues.

3D bioprinting has emerged as a promising technology in the field of tissue engineering and regenerative medicine due to its ability to create anatomically complex tissue substitutes. However, it still remains challenging to develop bioactive bioinks that provide appropriate and permissive environments to instruct and guide the regenerative process in vitro and in vivo. In this study alginate sulfate, a sulfated glycosaminoglycan (sGAG) mimic, was used to functionalize an alginate-gelatin methacryloyl (GelMA) interpenetrating network (IPN) bioink to enable the bioprinting of cartilaginous tissues. The inclusion of alginate sulfate had a limited influence on the viscosity, shear-thinning and thixotropic properties of the IPN bioink, enabling high-fidelity bioprinting and supporting mesenchymal stem cell (MSC) viability post-printing. The stiffness of printed IPN constructs greatly exceeded that achieved by printing alginate or GelMA alone, while maintaining resilience and toughness. Furthermore, given the high affinity of alginate sulfate to heparin-binding growth factors, the sulfated IPN bioink supported the sustained release of transforming growth factor-ß3 (TGF-ß3), providing an environment that supported robust chondrogenesis in vitro, with little evidence of hypertrophy or mineralization over extended culture periods. Such bioprinted constructs also supported chondrogenesis in vivo, with the controlled release of TGF-ß3 promoting significantly higher levels of cartilage-specific extracellular matrix deposition. Altogether, these results demonstrate the potential of bioprinting sulfated bioinks as part of a 'single-stage' or 'point-of-care' strategy for regenerating cartilaginous tissues. STATEMENT OF SIGNIFICANCE: This study highlights the potential of using sulfated interpenetrating network (IPN) bioink to support the regeneration of phenotypically stable articular cartilage. Construction of interpenetrating networks in the bioink enables unique high-fidelity bioprinting and provides synergistic increases in mechanical properties. The presence of alginate sulfate enables the capacity of high affinity-binding of TGF-ß3, which promoted robust chondrogenesis in vitro and in vivo.

3D bioprinting of prevascularised implants for the repair of critically-sized bone defects.

For 3D bioprinted tissues to be scaled-up to clinically relevant sizes, effective prevascularisation strategies are required to provide the necessary nutrients for normal metabolism and to remove associated waste by-products. The aim of this study was to develop a bioprinting strategy to engineer prevascularised tissues in vitro and to investigate the capacity of such constructs to enhance the vascularisation and regeneration of large bone defects in vivo. From a screen of different bioinks, a fibrin-based hydrogel was found to best support human umbilical vein endothelial cell (HUVEC) sprouting and the establishment of a microvessel network. When this bioink was combined with HUVECs and supporting human bone marrow stem/stromal cells (hBMSCs), these microvessel networks persisted in vitro. Furthermore, only bioprinted tissues containing both HUVECs and hBMSCs, that were first allowed to mature in vitro, supported robust blood vessel development in vivo. To assess the therapeutic utility of this bioprinting strategy, these bioinks were used to prevascularise 3D printed polycaprolactone (PCL) scaffolds, which were subsequently implanted into critically-sized femoral bone defects in rats. Micro-computed tomography (µCT) angiography revealed increased levels of vascularisation in vivo, which correlated with higher levels of new bone formation. Such prevascularised constructs could be used to enhance the vascularisation of a range of large tissue defects, forming the basis of multiple new bioprinted therapeutics. STATEMENT OF SIGNIFICANCE: This paper demonstrates a versatile 3D bioprinting technique to improve the vascularisation of tissue engineered constructs and further demonstrates how this method can be incorporated into a bone tissue engineering strategy to improve vascularisation in a rat femoral defect model.

Biofabrication and bioprinting using cellular aggregates, microtissues and organoids for the engineering of musculoskeletal tissues.

The modest clinical impact of musculoskeletal tissue engineering (TE) can be attributed, at least in part, to a failure to recapitulate the structure, composition and functional properties of the target tissue. This has motivated increased interest in developmentally inspired TE strategies, which seek to recapitulate key events that occur during embryonic and post-natal development, as a means of generating truly biomimetic grafts to replace or regenerate damaged tissues and organs. Such TE strategies can be substantially enabled by emerging biofabrication and bioprinting strategies, and in particular the use of cellular aggregates, microtissues and organoids as 'building blocks' for the development of larger tissues and/or organ precursors. Here, the application of such biological building blocks for the engineering of musculoskeletal tissues, from vascularised bone to zonally organised articular cartilage, will be reviewed. The importance of first scaling-down to later scale-up will be discussed, as this is viewed as a key component of engineering functional grafts using cellular aggregates or microtissues. In the context of engineering anatomically accurate tissues of scale suitable for tissue engineering and regenerative medicine applications, novel bioprinting modalities and their application in controlling the process by which cellular aggregates or microtissues fuse and self-organise will be reviewed. Throughout the paper, we will highlight some of the key challenges facing this emerging field. STATEMENT OF SIGNIFICANCE: The field of bioprinting has grown substantially in recent years, but despite the hype and excitement it has generated, there are relatively few examples of bioprinting strategies producing implants with superior regenerative potential to that achievable with more traditional tissue engineering approaches. This paper provides an up-to-date review of emerging biofabrication and bioprinting strategies which use cellular aggregates and microtissues as 'building blocks' for the development of larger musculoskeletal tissues and/or organ precursors - a field of research that can potentially enable functional regeneration of damaged and diseased tissues. The application of cellular aggregates and microtissues for the engineering of musculoskeletal tissues, from vascularised bone to zonally organised articular cartilage, will be reviewed. In the context of engineering anatomically accurate tissues of scale, novel bioprinting modalities and their application in controlling the process by which cellular aggregates or microtissues self-organise is addressed, as well as key challenges facing this emerging field.

Development of a New Bone-Mimetic Surface Treatment Platform: Nanoneedle Hydroxyapatite (nnHA) Coating.

The hierarchical structure of bone plays pivotal roles in driving cell behavior and tissue regeneration and must be considered when designing materials for orthopedic applications. Herein, it is aimed to recapitulate the native bone environment by using melt electrowriting to fabricate fibrous microarchitectures which are modified with plate-shaped (pHA) or novel nanoneedle-shaped (nnHA) crystals. Nuclear magnetic resonance spectroscopy, scanning electron microscopy, transmission electron microscopy, and X-ray diffraction demonstrate that these coatings replicate the nanostructure and composition of native bone. Human mesenchymal stem/stromal cell (MSC) mineralization is significantly increased fivefold with pHA scaffolds and 14-fold with nnHA scaffolds. Given the protein stabilizing properties of mineral, these materials are further functionalized with bone morphogenetic protein 2 (BMP2). nnHA treatment facilitates controlled release of BMP2 which further enhance MSC mineral deposition. Finally, the versatility of this nnHA treatment method, which may be used to coat different architectures/materials including fused deposition modeling (FDM) scaffolds and Ti6Al4V titanium, is demonstrated. This study thus outlines a method for fabricating scaffolds with precise fibrous microarchitectures and bone-mimetic nnHA extrafibrillar coatings which significantly enhance MSC osteogenesis and therapeutic protein delivery, and leverages these results to show how this surface treatment method may be applied to a wider field for multiple orthopedic applications.